effects of hmw- & lmw-glutenins and grain hardness on size of wheat storage proteins polymers
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Effects of HMW- & LMW-glutenins and grain hardness on size of wheat
storage proteins polymers
Véronique S. LESAGE, L. RHAZI, T. AUSSENAC, B. MELEARD, G. BRANLARD
INRA, Genetics, Diversity and Ecophysiology of Cereals, Clermont-Ferrand, France
11th International Gluten Workshop, August 12-15, 2012
Dough quality
Nature of gliadins and glutenins alleles
Polymerization
LMW-Glutenin
g-gliadin
PS N Repeat C
From Köhler P. et al., 1993. Z. Lebensm. Unters. Forsch
LMW-Glu PS N Repeat C
HMW-GS type y PS Repeat
C N
PS N Repeat C
PS Repeat
C N HMW-GS type y
LMW-Glu
Cys: intermolecular disulfide bonds
Cys: intramolecular disulfide bonds
HMW-GS type x PS
Repeat C N
Polymers size depends on free cystine number and also on environmental conditions
Polymerization/aggregation
Formation of insoluble polymers during wheat kernel development
Source Carceller JL, Aussenac T., 2001. Aust. J. Plant Physiol
From Lesage et al., 2011, J. Cereal Science, 53(2), 231
0
0.5
1
1.5
2
2.5
3
3.5
4
1997 2008 2010
Mw
x 1
07
g/m
ol
hard
soft
NILs
Molecular weight X 106 Da
PinA
PinA x
40 35
30
25
20
15
10
5
0
Influence of puroindolines and glutenins alleles
on polymers size?
Material and
methods
Material and methods
2009: L1 L2 L3
2010: L4 L5 L6
Material
Methods / Investigated traits HMW- and LMW- Glutenin alleles (SDS-PAGE) Grain hardness (NIRS) Polymer mass (Mw) and radius of giration (Rw) Polydispersity index (Mw/Mn) % protein fractions (SE-HPLC) protein content (NIRS)
puroindolines alleles (sequencing)
AFFFF
68 bread-wheat varieties 2 years 3 locations
(L1 –L6)
Methods. Asymmetrical flow field-flow fractionation
AFFFF-MALLS analytical device used for molecular characterization of storage proteins
(Multi Angular Laser Light Scattering)
Principle
Methods. Asymmetrical flow field-flow fractionation
30 mg flour 1 ml sodium phosphate buffer 0.1M, pH 6.9 2% SDS
15 sec. sonication 20 W
60°C – 15 min
Sample preparation
Methods. Asymmetrical flow field-flow fractionation
(Multi Angular Laser Light Scattering detector)
Polymers size and conformation
MALLS chromatogram
Polymers Monomers
Methods. Asymmetrical flow field-flow fractionation
Measurements: - Mw: weight-average - Mn: number-average - Mw/Mn: polydispersity index - Rw: weight-average mean square radius (radius of giration)
Methods. Size Exclusion - HPLC
Sample preparation
160 mg flour 20 ml sodium phosphate buffer 0.1M 1% SDS
60°C – 80 min stirring
3 min sonication 2 W
filtration 0.2 µm
TSK gel G4000sw Silica-based, pore size: 13-17 µm
SE-HPLC
5 protein fractions (F1- F5)
F1 F2
F4
F3
F5
Elution Time
Large Glu Pol.
Small Glu Pol.
Ω- Gli
γ,β- Gli
α-Gli, Alb, Glo.
Results
Variation of polymers characteristics
AFFFF
Mw 2
Number
0 1 2 3 4 5(X 1,E7)
0
10
20
30
40
50
60
Mw2 Mw /Mn2
Number
0 20 40 60 80 100
0
20
40
60
80
Mw/Mn2 Rw 2 (nm)
Number
20 40 60 80 100 120 140
0
10
20
30
40
Rw2 (nm)
SE-HPLC
SE-HPLC F1 %
Number
10 12 14 16 18
0
10
20
30
40
50
SE-HPLC F2 %
Number
17 19 21 23 25 27 29
0
10
20
30
40
SE-HPLC F3 %
Number
5,9 6,9 7,9 8,9 9,9
0
5
10
15
20
25
30
Mean F1 : 14% Mean F2 : 24% Mean F3 : 7%
5.4 - 48.8 million Da
0.75 - 2 million Da 90.000 – 750.000 Da 50.000 – 90.000 Da
36.5 – 116.2 nm 7.1 – 82.9
(Polymers mass) (Polydispersity index) (Polymers radius)
Glutenin loci effects on quality traits
Protein content
Gluten Index
Grain Hardness
Mw2 Mw/Mn2 Rw2 %F1 %F2 %F3 %F4 %F5
Glu-A1 ns ns ns ns ns ns ns ns ns ns ns
HMW- Glu
Glu-B1 ns ns ns * ** * * * ns ns ns
Glu-D1 ns *** *** ns ns ns *** *** * ns ns
Glu-A3 ns ns * ns ** ns *** ns ** *** ns
LMW- Glu
Glu-B3 ns ns ns ns ns ns *** *** *** *** **
Glu-D3 ns ns ns *** *** *** ns ns ns ns *
R2 0.2 17.0 19.9 9.0 15.9 11.2 47.1 58.7 16.9 45.3 10.5
Glutenin loci effects on quality traits
Protein content
Gluten Index
Grain Hardness
Mw2 Mw/Mn2 Rw2 %F1 %F2 %F3 %F4 %F5
Glu-A1 ns ns ns ns ns ns ns ns ns ns ns
HMW- Glu
Glu-B1 ns ns ns * ** * * * ns ns ns
Glu-D1 ns *** *** ns ns ns *** *** * ns ns
Glu-A3 ns ns * ns ** ns *** ns ** *** ns
LMW- Glu
Glu-B3 ns ns ns ns ns ns *** *** *** *** **
Glu-D3 ns ns ns *** *** *** ns ns ns ns *
R2 0.2 17.0 19.9 9.0 15.9 11.2 47.1 58.7 16.9 45.3 10.5
Effects of Glutenins alleles on polymers mass
Standardized coef.
-0,16
-0,06
0,04
0,14
0,24
Glu
A1-
nG
luA
1-1
Glu
A1-
2*
Glu
B1-
68
Glu
B1-
7G
luB
1-7
8G
luB
1-7
9G
luB
1-1
316
Glu
B1-
141
5G
luB
1-1
718
Glu
B1-
6.1
-22
Glu
D1-
212
Glu
D1-
312
Glu
D1-
412
Glu
D1-
510
Glu
A3-
aG
luA
3-e
fG
luA
3-d
Glu
B3-
bG
luB
3-b
pG
luB
3-c
Glu
B3-
cp
Glu
B3-
dG
luB
3-f
Glu
B3-
gG
luB
3-j
Glu
D3-
bG
luD
3-c
Mw2
Mw2
- 0.16
- 0.06
0.04
0.14
0.24
Standardized coef.
Glu-B1 Glu-D1 Glu-A3 Glu-B3 Glu-D3
B1 (7)
D3 (b)
Glu-A1
Puroindoline B alleles
• 3 Grain Hardness classes: Soft < 35
35 < Medium < 75
Hard > 75
• PinA and PinB genes sequenced in all varieties
– PinA: no deletion, no SNP
– PinB : 5 alleles
PinB-D1a WPTKWWKGGC-----------L
PinB-D1b WPTKWWKSGC-----------L
PinB-D1d WPTKWRKGGC-----------L
PinB-D1c WPTKWWKGGC-----------P
PinB-D1b + d WPTKWRKSGC-----------L
AA position from start: 73 75 89
Allele of Soft varieties
Puroindoline B alleles
• 3 Grain Hardness classes: Soft < 35
35 < Medium < 75
Hard > 75
• PinA and PinB genes sequenced in all varieties
– PinA: no deletion, no SNP
– PinB : 5 alleles
Occurence
Grain Hardness Class
0 20 40 60 80
S
M
H
PinBD1aD1bD1dD1cD1b+dPinB-D1a WPTKWWKGGC-----------L
PinB-D1b WPTKWWKSGC-----------L
PinB-D1d WPTKWRKGGC-----------L
PinB-D1c WPTKWWKGGC-----------P
PinB-D1b + d WPTKWRKSGC-----------L
Effects of interaction between glutenins and puroindoline B alleles
Grain Hardness Mw2 Mw/Mn2 Rw2 %F3
Glu Glu x PinB Glu Glu x PinB Glu Glu x PinB Glu Glu x PinB Glu Glu x PinB
effect effect R2 effect effect R2 effect effect R2 effect effect R2 effect effect R2
A1 ns ns ** 15 ns ns ns * 15 ns ns
HMW-Glu
B1 ns * ** 14 ** ** 18 * ** 14 ns * 23
D1 *** *** 27.7 ns ns ns ns ns * 15 * ** 18
A3 * ns * 13 ** *** 22 ns ns ** *** 19
LMW-Glu
B3 ns ns ns ns ns ns ns *** *** 20
D3 ns *** ns *** * 11 *** ns ns * 18
R2 19.9 9.0 15.9 11.2 16.8
Effects of interaction between glutenins and puroindoline B alleles
Grain Hardness Mw2 Mw/Mn2 Rw2 %F3
Glu Glu x PinB Glu Glu x PinB Glu Glu x PinB Glu Glu x PinB Glu Glu x PinB
effect effect R2 effect effect R2 effect effect R2 effect effect R2 effect effect R2
A1 ns ns ** 15 ns ns ns * 15 ns ns
HMW-Glu
B1 ns * ** 14 ** ** 18 * ** 14 ns * 23
D1 *** *** 27.7 ns ns ns ns ns * 15 * ** 18
A3 * ns * 13 ** *** 22 ns ns ** *** 19
LMW-Glu
B3 ns ns ns ns ns ns ns *** *** 20
D3 ns *** ns *** * 11 *** ns ns * 18
R2 19.9 9.0 15.9 11.2 16.8
Effects of interaction between glutenins and puroindoline B alleles
Glu-B1
-1
4
9
14
19
24
29(X 1,E6)
Mw2
7 68 78 79 1316 1718 6122
Pinb-3alD1aD1bD1d
PinB
Mw2
Glu-B1
PinB ___ D1a ___ D1b ___ D1d
4 9
14
19 24
29 x 106 Da
7-8 Glu-A3
13
15
17
19
21(X 1,E6)
Mw2
a d ef
Pinb-3alD1aD1bD1d
PinB
Mw2
Glu-A3
PinB ___ D1a ___ D1b ___ D1d
x 106 Da
13
15
17
19
21
a
Interactions Glutenins-Puroindolines had effects on polymers characteristics Effects were contrasted depending on each allele Small part of polymers characteristics variation
Part of environmental effects?
Environmental effects on quality traits
Year effect R2
Protein content ns
Grain Hardness ns
Gluten Index ns
Mw2 * 44.9
Mw/Mn2 * 51.0
Rw2 ** 56.4
%F1 ns
%F2 ns
%F3 ns
%F4 ns
%F5 ns
* p < 0.05
** p < 0.01
*** p < 0.001
Variance analysis (68 varieties, 2 years)
%
Effects of sum of mean temperatures (June and July)
effect R2
Protein content ns
Grain Hardness ns
Gluten Index ns Mw2 *** 38.5 Mw/Mn2 *** 62.7
Rw2 *** 47.5 %F1 ns
%F2 ns %F3 ns
%F4 ns
%F5 ns
%
Environmental effects on quality traits
SMT-Jun Ju l (癈 )
1080 1100 1120 1140 1160 1180
0
1
2
3
4
5(X 1,E7)
Mw2 Mw2
Sum of mean temperatures (°C) (June and July)
1080 1100 1120 1140 1160 1180
0
10
20
30
40
50
x 106 Da
AFFFF gives more reliable data for native polymers characterization than SE-HPLC that reflects more diversity of glutenins
Regression Mw2 on sum of daily mean temperatures of June and July
Soft : Mw2= -203207000+196542*SumTemp R²=0.37 Medium : Mw2= -172673000+167021*SumTemp R²=0.48 Hard : Mw2= -124367000+121462*SumTemp R²=0.44
(°C)
Mw2
Sum mean temp.
0.00E+00
5.00E+06
1.00E+07
1.50E+07
2.00E+07
2.50E+07
3.00E+07
3.50E+07
1080 1100 1120 1140 1160 1180 1200 1220
Soft
Medium
Hard
ns
ns
p < 0.01
35
x 106 Da
30
25
20
15
10
5
0
**
S
H
1080 1100 1120 1140 1160 1180 1200 1220
Temperature had a different impact on polymers size according to grain hardness and this has probably a strong impact on dough quality
Conclusions
Conclusions
• AFFFF provides more reliable data on polymers size than SE-HPLC
• Influence of PinB alleles on storage proteins polymers was observed in interaction with glutenins alleles
• Temperatures during the 2 last months impacted characteristics of polymers differently according to grain hardness
Acknowledgements
Private partners: Saaten Union Research Syngenta Momont Florimond-Desprez Caussade semences
Gérard Branlard François-Xavier Oury
Larbi Rhazi Thierry Aussenac
Benoît Méléard
Thank you
Glu-A1 0 1 2*
HMW Glu-B1 6-8 7 7-8 7-9 13-16 14-15 17-18 6.1-22
Glu-D1 2-12 3-12 4-12 5-10
Glu-A3 a d ef
LMW Glu-B3 b b' c c' d f g j
Glu-D3 b c
Glutenins alleles in the experiment
Falcon Hard-Soft NILs + equal Temp.
• Soft (PinA +, PinB +)
Mw2 : 20.4 106 Da
• Hard (PinA -, PinB +)
Mw2 : 28.5 106 Da
Hypothesis:
PinA absence increased aggregation
68 varieties + different Temp.
• Soft (PinA +, PinB +)
Average Mw2 : 17.6 106 Da
• Hard (PinA +, PinB-SNPs)
Average Mw2 : 13.0 106 Da
Hypothesis:
SNP-PinB decreased polymerization
Comparison of Mw2 in the 2 experiments
From Lesage et al., 2011. J. of Cereal Science and Lesage et al., 2012, J. Exp. Bot
From this experiment
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